Cik M, Chazot P L, Stephenson F A
Department of Pharmaceutical Chemistry, School of Pharmacy, London, U.K.
Biochem J. 1993 Dec 15;296 ( Pt 3)(Pt 3):877-83. doi: 10.1042/bj2960877.
The N-methyl-D-aspartate R1 (NMDAR1) and NMDAR2A subunits were expressed transiently either alone or in combination in human embryonic kidney (HEK) 293 cells. The biochemical and pharmacological properties of the cloned receptors were compared with those of adult rat brain NMDA receptors using both immunological methods with a newly developed anti-NMDAR2A-(1435-1445) antibody and [3H]MK801 radioligand binding activity. Anti-NMDAR2A-(1435-1445) antibodies recognized specifically four immunoreactive species with M(r)s of 180,000, 122,000, 97,000 and 54,000 in rat brain, but only a single band of M(r) 180,000 in HEK 293 cells singly transfected with plasmid pCISNMDAR2A. N-deglycosylation of HEK cell membranes yielded a 165,000-M(r) immunoreactive species, which is in agreement with the size predicted from the cDNA sequence for the mature NMDAR2A subunit. Co-expression of NMDAR1 and NMDAR2A subunits in HEK 293 cells resulted in cell death. Thus conditions were established for the optimum expression of heteromeric receptors in viable cells, including a requirement for DL-2-amino-5-phosphonopentanoic acid (AP5) in the culture medium post-transfection. Cells transfected with pCISNMDAR1 and pCISNMDAR2A combined yielded a 10-fold increase in the number of [3H]MK801 binding sites compared with single subunit expression. MK801 had similar affinity for the expressed receptors as for those found in adult rat and mouse brain. These results demonstrate that the NMDAR1 and NMDAR2A receptor subunits co-assemble to form a heteromeric complex with properties similar to those of the native receptors of adult mammalian forebrain. Furthermore, the conditions reported for maximal transient expression provide a basis for further structure-activity studies.
N-甲基-D-天冬氨酸受体1（NMDAR1）和NMDAR2A亚基单独或联合在人胚肾（HEK）293细胞中瞬时表达。使用新开发的抗NMDAR2A-(1435 - 1445)抗体的免疫方法和[3H]MK801放射性配体结合活性，将克隆受体的生化和药理学特性与成年大鼠脑NMDA受体的特性进行比较。抗NMDAR2A-(1435 - 1445)抗体在大鼠脑中特异性识别出四种免疫反应性条带，其分子量分别为180,000、122,000、97,000和54,000，但在单独用质粒pCISNMDAR2A转染的HEK 293细胞中仅识别出一条分子量为180,000的条带。HEK细胞膜的N-去糖基化产生了一个分子量为165,000的免疫反应性条带，这与成熟NMDAR2A亚基的cDNA序列预测的大小一致。NMDAR1和NMDAR2A亚基在HEK 293细胞中的共表达导致细胞死亡。因此，建立了在活细胞中最佳表达异聚体受体的条件，包括转染后培养基中需要DL-2-氨基-5-磷酸戊酸（AP5）。与单个亚基表达相比，用pCISNMDAR1和pCISNMDAR2A联合转染的细胞产生的[3H]MK801结合位点数量增加了10倍。MK801对表达的受体的亲和力与成年大鼠和小鼠脑中发现的受体相似。这些结果表明，NMDAR1和NMDAR2A受体亚基共同组装形成一个异聚体复合物，其特性与成年哺乳动物前脑的天然受体相似。此外，所报道的最大瞬时表达条件为进一步的构效关系研究提供了基础。
